Mara D'Onofrio

Familial basilar migraine associated with a new mutation in the ATP1A2 gene

Basilar migraine (BM), familial hemiplegic migraine (FHM), and sporadic hemiplegic migraine (SHM) are phenotypically similar subtypes of migraine with aura, differentiated only by motor symptoms, which are absent in BM. Mutations in CACNA1A and ATP1A2 have been found in FHM. The authors detected a novel mutation in the ATP1A2 gene (R548H) in members of a family with BM, suggesting that BM and FHM may be allelic disorders.

Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer.

The human intestinal Caco‐2 cell line has been extensively used as a model of the intestinal barrier. However, it is widely reported in literature that culture‐related conditions, as well as the different Caco‐2 cell lines utilized in different laboratories, often lead to problems of reproducibility making difficult to compare results. We developed a new cell‐maintenance protocol in which Caco‐2 cells were subcultured at 50% of confluence instead of 80% of confluence, as usually suggested. Using this new protocol, Caco‐2 cells retained a higher proliferation potential resulting in a cell population, which, on reaching confluence, was able to differentiate almost synchronously, forming a more homogeneous and polarized cell monolayer, as compared to that obtained using a high cell growing density. This comparison has been done by analyzing the gene expression and the structural characteristics of the 21‐days differentiated monolayers by microarrays hybridization and by confocal microscopy. We then investigated if these differences could also modify the effects of toxicants on 21‐days‐differentiated cells. We analyzed the 2 h‐acute toxicity of CuCl2 in terms of actin depolymerization and metallothionein 2A (MT2A) and heat shock protein 70 (HSPA1A) genes induction. Copper treatment resulted in different levels of actin depolymerization and gene expression induction in relationship with culture protocol, the low‐density growing cells showing a more homogeneous and stronger response. Our results suggest that cell growing density could influence a number of morphological and physiological properties of differentiated Caco‐2 cells and these effects must be taken in account when these cells are used as intestinal model. J. Cell. Physiol. 226: 1531–1543, 2011.

Loss of TIMP3 underlies diabetic nephropathy via FoxO1/STAT1 interplay.

ADAM17 and its inhibitor TIMP3 are involved in nephropathy, but their role in diabetic kidney disease (DKD) is unclear. Diabetic Timp3−/− mice showed increased albuminuria, increased membrane thickness and mesangial expansion. Microarray profiling uncovered a significant reduction of Foxo1 expression in diabetic Timp3−/− mice compared to WT, along with FoxO1 target genes involved in autophagy, while STAT1, a repressor of FoxO1 transcription, was increased. Re‐expression of Timp3 in Timp3−/− mesangial cells rescued the expression of Foxo1 and its targets, and decreased STAT1 expression to control levels; abolishing STAT1 expression led to a rescue of FoxO1, evoking a role of STAT1 in linking Timp3 deficiency to FoxO1. Studies on kidney biopsies from patients with diabetic nephropathy confirmed a significant reduction in TIMP3, FoxO1 and FoxO1 target genes involved in autophagy compared to controls, while STAT1 expression was strongly increased.

In the Adult Hippocampus, Chronic Nerve Growth Factor Deprivation Shifts GABAergic Signaling from the Hyperpolarizing to the Depolarizing Direction

GABA, the main inhibitory transmitter in adulthood, early in postnatal development exerts a depolarizing and excitatory action. This effect, which results from a high intracellular chloride concentration ([Cl−]i), promotes neuronal growth and synaptogenesis. During the second postnatal week, the developmental regulated expression of the cation-chloride cotransporter KCC2 accounts for the shift of GABA from the depolarizing to the hyperpolarizing direction. Changes in chloride homeostasis associated with high [Cl−]i have been found in several neurological disorders, including temporal lobe epilepsy. Here, we report that, in adult transgenic mice engineered to express recombinant neutralizing anti-nerve growth factor antibodies (AD11 mice), GABA became depolarizing and excitatory. AD11 mice exhibit a severe deficit of the cholinergic function associated with an age-dependent progressive neurodegenerative pathology resembling that observed in Alzheimer patients. Thus, in hippocampal slices obtained from 6-month-old AD11 (but not wild-type) mice, the GABAA agonist isoguvacine significantly increased the firing of CA1 principal cells and, at the network level, the frequency of multiunit activity recorded with extracellular electrodes. In addition, in AD11 mice, the reversal of GABAA-mediated postsynaptic currents and of GABA-evoked single-channel currents were positive with respect to the resting membrane potential as estimated in perforated patch and cell attached recordings, respectively. Real-time quantitative reverse transcription-PCR and immunocytochemical experiments revealed a reduced expression of mRNA encoding for Kcc2 and of the respective protein. This novel mechanism may represent a homeostatic response that counterbalances within the hippocampal network the Alzheimer-like neurodegenerative pathology found in AD11 mice.

Gene Expression Changes in the Motor Cortex Mediating Motor Skill Learning

The primary motor cortex (M1) supports motor skill learning, yet little is known about the genes that contribute to motor cortical plasticity. Such knowledge could identify candidate molecules whose targeting might enable a new understanding of motor cortical functions, and provide new drug targets for the treatment of diseases which impair motor function, such as ischemic stroke. Here, we assess changes in the motor-cortical transcriptome across different stages of motor skill acquisition. Adult rats were trained on a gradually acquired appetitive reach and grasp task that required different strategies for successful pellet retrieval, or a sham version of the task in which the rats received pellet reward without needing to develop the reach and grasp skill. Tissue was harvested from the forelimb motor-cortical area either before training commenced, prior to the initial rise in task performance, or at peak performance. Differential classes of gene expression were observed at the time point immediately preceding motor task improvement. Functional clustering revealed that gene expression changes were related to the synapse, development, intracellular signaling, and the fibroblast growth factor (FGF) family, with many modulated genes known to regulate synaptic plasticity, synaptogenesis, and cytoskeletal dynamics. The modulated expression of synaptic genes likely reflects ongoing network reorganization from commencement of training till the point of task improvement, suggesting that motor performance improves only after sufficient modifications in the cortical circuitry have accumulated. The regulated FGF-related genes may together contribute to M1 remodeling through their roles in synaptic growth and maturation.

Assessing the relative incidence of mitochondrial DNA A3243G in migraine without aura with maternal inheritance

Objective.—To determine whether patients with migraine without aura with maternal “inheritance” are affected by a monosymptomatic form of the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes) or carry the most common mitochondrial DNA (mtDNA) mutation associated with MELAS, namely the A3243G transition in the transfer RNA (tRNA)Leu(UUR) gene.

mtDNA A3243G MELAS mutation is not associated with multigenerational female migraine

Article abstract The authors searched for mitochondrial DNA (mtDNA) A3243G mutation in peripheral blood leukocytes from female migraine patients with pure matrilinear history of migraine along two or three generations. The current study was designed to exclude any male transmission of the disease. The mutation was absent in all patients. We conclude that mtDNA A3243G mutation does not contribute to the pathogenesis of pure matrilinear multigenerational migraine with or without aura.

The interplay of two single nucleotide polymorphisms in the CACNA1A gene may contribute to migraine susceptibility

Migraine is a common disorder with a significant genetic component. Mutations in the CACNA1A gene are found in hemiplegic migraine (HM). Basilar-type (BM), another subtype of migraine with aura, differs from HM only by the absence of motor deficits. BM and HM may thus share common genetic features. In the present study, two single nucleotide polymorphisms (SNPs) of the CACNA1A gene were characterized in a population of migraine patients and healthy controls. The polymorphisms, E918D, predicting a glutamic acid-to-aspartic acid substitution at codon 918 and E993V, predicting a glutamic acid-to-valine substitution at codon 993, were frequently detected among patients and controls. Seven BM, 10 SHM, 5 FHM, 57 migraine with typical aura, 32 migraine without aura patients and 107 healthy controls were screened. The E918D and E993V SNPs were found in 30/117 (25.6%) and 32/117 (27.3%) migraine patients, respectively. The prevalence of these SNPs taken separately was not significantly different from that of control subjects (n=28/107, 26.2% for E918D; n=29/107 for E993V, 27.1%) neither for the total migraine population nor for the various migraine subtypes. By contrast, coexistence of both SNPs was more frequent in migraineurs (25/117, 21%) than in healthy controls (12/107, 11%; p=0.048), a difference that was significant for every migraine subtype. This result suggests that the interplay of minor genetic variants such as single nucleotide polymorphisms may influence the P/Q-type calcium channel function in several subtypes of migraine.

Targeting the MDM2/MDM4 interaction interface as a promising approach for p53 reactivation therapy

Restoration of wild-type p53 tumor suppressor function has emerged as an attractive anticancer strategy. Therapeutics targeting the two p53-negative regulators, MDM2 and MDM4, have been developed, but most agents selectively target the ability of only one of these molecules to interact with p53, leaving the other free to operate. Therefore, we developed a method that targets the activity of MDM2 and MDM4 simultaneously based on recent studies indicating that formation of MDM2/MDM4 heterodimer complexes are required for efficient inactivation of p53 function. Using computational and mutagenesis analyses of the heterodimer binding interface, we identified a peptide that mimics the MDM4 C-terminus, competes with endogenous MDM4 for MDM2 binding, and activates p53 function. This peptide induces p53-dependent apoptosis in vitro and reduces tumor growth in vivo. Interestingly, interfering with the MDM2/MDM4 heterodimer specifically activates a p53-dependent oxidative stress response. Consistently, distinct subcellular pools of MDM2/MDM4 complexes were differentially sensitive to the peptide; nuclear MDM2/MDM4 complexes were particularly highly susceptible to the peptide-displacement activity. Taken together, these data identify the MDM2/MDM4 interaction interface as a valuable molecular target for therapeutic reactivation of p53 oncosuppressive function.

NGF and proNGF regulate functionally distinct mRNAs in PC12 cells: an early gene expression profiling.

The biological activities of NGF and of its precursor proNGF are quite distinct, due to different receptor binding profiles, but little is known about how proNGF regulates gene expression. Whether proNGF is a purely pro-apoptotic molecule and/or simply a “less potent NGF” is still a matter of debate. We performed experiments to address this question, by verifying whether a proNGF specific transcriptional signature, distinct from that of NGF, could be identified. To this aim, we studied gene expression regulation by proNGF and NGF in PC12 cells incubated for 1 and 4 hours with recombinant NGF and proNGF, in its wild-type or in a furin-cleavage resistant form. mRNA expression profiles were analyzed by whole genome microarrays at early time points, in order to identify specific profiles of NGF and proNGF. Clear differences between the mRNA profiles modulated by the three neurotrophin forms were identified. NGF and proNGF modulate remarkably distinct mRNA expression patterns, with the gene expression profile regulated by NGF being significantly more complex than that by proNGF, both in terms of the total number of differentially expressed mRNAs and of the gene families involved. Moreover, while the total number of genes modulated by NGF increases dramatically with time, that by proNGFs is unchanged or reduced. We identified a subset of regulated genes that could be ascribed to a “pure proNGF” signalling, distinct from the “pure NGF” one. We also conclude that the composition of mixed NGF and proNGF samples, when the two proteins coexist, influences the profile of gene expression. Based on this comparison of the gene expression profiles regulated by NGF and its proNGF precursor, we conclude that the two proteins activate largely distinct transcriptional programs and that the ratio of NGF to proNGF in vivo can profoundly influence the pattern of regulated mRNAs.